Roles Of PI3K/AKT And NF-κB Pathways On Oxidative Damage And Inflammation Induced By Low Molecular Weight-PAHs In A549 Cells

Objectives: Polycyclic aromatic hydrocarbons（PAHs）are widespread organic pollutants in the environment,which may lead to a variety of respiratory diseases.In this study,Phenanthrene（Phe）and fluorene（Flu）individually and as binary PAH mixture were chosen as the target subjects and their impacts on oxidative stress,inflammatory response,and PI3K/AKT and NF-κB signaling pathways were detected in A549 cells to explore the potential mechanisms of lung damage.Methods:（1）Cell viability was firstly detected by MTT method,and A549 cells were exposed to various concentrations（200-800 μM）of Phe,Flu and their binary mixture for 24 and 48 hours to determine the appropriate exposure dose and time;（2）The contents of reactive oxygen species（ROS）,malondialdehyde（MDA）and the activities of superoxide dismutase（SOD）and catalase（CAT）were detected by biochemical kits to evaluate oxidative damage;（3）The protein and gene expression levels of intracellular interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）,transforming growth factor-β（TGF-β）and the protein content of alveolar surface-associated protein A（SP-A）were measured by Western blot and RT-q PCR methods to assess the inflammatory response;（4）The total protein expression levels and phosphorylation levels of PI3K、AKT、IκBα、NF-κB p65 of PI3K/AKT and NF-κB signaling pathways were detected,and the protein expression levels of inflammatory factors after pretreatment with PI3K/AKT and NF-κB inhibitors（LY294002 and BAY11-7082）were measured by Western blot method to assess the possible molecular mechanism of inflammatory response.Results:（1）The MTT results showed that the cell viability was decreased with the increase of time and the concentration of Phe,Flu and their mixture.After treatment with 200～800 μM Phe,Flu and Phe+Flu for 24 hours,compared with the control group,the cell viability was decreased significently among each group,except for 200 μM Phe group（P<0.05）,and the cell viability was decreased significantly in all dose groups after treating with Phe,Flu and Phe+Flu for 48 hours（P<0.05）.（2）Based on cell viability experiments,200,400 and 600 μM were selected as the trial dose and the toxic effects of LMW-PAHs on A549 cells were observed after treating for 24 hours.The results showed that 600 μM Phe,flu and their binary mixture of Phe and flu could induce excessive productions of ROS,and the content of ROS in the combined exposure group was significantly higher than that in the single group（P<0.05）.The contents of MDA increased significantly in 600 μM Phe group,and in 400 μM and 600 μM Flu and Phe+Flu groups（P<0.05）.The activities of SOD increased significantly in 600 Phe,Flu groups and 400 and 600 Phe+Flu groups（P<0.05）.The activities of CAT increased significantly in 600 μM Phe,Flu groups,and in each group of Phe+Flu（P<0.05）.（3）Western blot results showed that compared with the control group,the protein expression levels of TNF-α increased significantly at 400 μM and 600 μM groups after treating with Phe,Flu and Phe+Flu,respectively（P<0.05）.And the protein expression levels of TNF-α in 400 μM co-exposed group was significantly higher than that in single exposed groups（P<0.05）.The protein expression levels of IL-6increased significantly at 600 μM group after treating with Phe and Flu,and increased at 400 and 600 μM groups after treating with Phe+Flu（P<0.05）.And the protein expression levels of IL-6 in 400 and 600 μM co-exposed group were significantly higher than that in single exposed groups（P<0.05）.There were no significant changes of TGF-β protein expressions in Phe,Flu and Phe+Flu groups.The protein expressions of SP-A were raised in 400 and 600 μM groups by treating with Phe and Flu,separately（P<0.05）,and increased in 200 and 400 μM groups after treatment with Phe+Flu,while it was decreased in 600 μM group when compared with 400 μM group,and significantly less than that of the single exposed group（P<0.05）.（4）RT-qPCR results showed that compared with the control group,the mRNA expression levels of TNF-α increased significantly at 600 μM group after treating with Phe,and increased at 400 and 600 μM groups after treating with Flu and Phe+Flu（P<0.05）.And the mRNA expression level of TNF-α in 400 μM co-exposed group was significantly higher than that in single Flu exposed group（P<0.05）.The mRNA expression levels of IL-6 increased significantly at 600 μM group after treating with Phe and Flu,and increased at 400 and 600 μM groups after treating with Phe+Flu（P<0.05）.There were no significant differences in the mRNA expressions of TGF-β in all concentrations of Phe,Flu,and their mixture（P>0.05）except for 400 μM Flu group.（5）The results of PI3K/AKT and NF-κB signaling pathways showed that there were no significant changes in the protein levels of PI3 k,AKT,NF-κB p65 in Phe,Flu and Phe+Flu groups（P>0.05）,and the protein levels of IκBα were also no significant changes in all treatment groups expect for 400 μM Flu group lower than Phe group.Compare with the control group,the phosphorylation levels of PI3 K were increased significantly at 400 and 600 μM Phe,600 μM Flu and 600 μM Phe+Flu groups（P<0.05）.The phosphorylation levels of AKT were increased significantly at 600 μM Flu,400 and 600 μM Phe and Phe+Flu groups（P<0.05）.The phosphorylation levels of IκBα were increased significantly at 600 μM Phe,600 μM Flu,400 and 600 μM Phe+Flu groups（P<0.05）.The phosphorylation levels of NF-κB were increased significantly at 600 μM Flu,400 and 600 μM Phe and Phe+Flu groups（P<0.05）.（6）The results of inhibitor test showed that compared with the control group,the phosphorylation levels of AKT and NF-κB p65 proteins were significantly increased after exposed to the mixture（P<0.05）.Pretreatment with LY294002 or BAY11-7082 significantly decreased the phosphorylation of AKT and NF-κB p65（P<0.05）.And pro-inflammatory cytokine expressions of TNF-α and IL-6 induced by the mixture were all alleviated by pretreatment with LY294002 or BAY11-7082（P<0.05）.Conclusion: The results demonstrated that Phe,Flu and their mixture could induce oxidative stress and subsequent inflammation in A549 cells,while combined inflammatory response was stronger than that of single action.PI3K/AKT and NF-κB signaling pathways played an important role in inflammation induced by LMW-PAHs.This study revealed a possible mechanism of lung damage induced by LMW-PAHs.